MicroRNA Biogenesis and Hedgehog-Patched Signaling ... - Genetics

HIGHLIGHTED ARTICLE | INVESTIGATION

MicroRNA Biogenesis and Hedgehog-Patched Signaling Cooperate to Regulate an Important Developmental Transition in Granule Cell Development Lena Constantin,*,1 Myrna Constantin,† and Brandon J. Wainwright*

*Institute for Molecular Bioscience and †Queensland Alliance for Agriculture and Food Innovation, University of Queensland, St. Lucia, Queensland 4072, Australia ORCID IDs: 0000-0003-1492-3845 (L.C.); 0000-0002-3287-4891 (M.C.)

ABSTRACT The Dicer1, Dcr-1 homolog (Drosophila) gene encodes a type III ribonuclease required for the canonical maturation and functioning of microRNAs (miRNAs). Subsets of miRNAs are known to regulate normal cerebellar granule cell development, in addition to the growth and progression of medulloblastoma, a neoplasm that often originates from granule cell precursors. Multiple independent studies have also demonstrated that deregulation of Sonic Hedgehog (Shh)-Patched (Ptch) signaling, through miRNAs, is causative of granule cell pathologies. In the present study, we investigated the genetic interplay between miRNA biogenesis and Shh-Ptch signaling in granule cells of the cerebellum by way of the Cre/lox recombination system in genetically engineered models of Mus musculus (mouse). We demonstrate that, although the miRNA biogenesis and Shh-Ptch-signaling pathways, respectively, regulate the opposing growth processes of cerebellar hypoplasia and hyperplasia leading to medulloblastoma, their concurrent deregulation was nonadditive and did not bring the growth phenotypes toward an expected equilibrium. Instead, mice developed either hypoplasia or medulloblastoma, but of a greater severity. Furthermore, some genotypes were bistable, whereby subsets of mice developed hypoplasia or medulloblastoma. This implies that miRNAs and Shh-Ptch signaling regulate an important developmental transition in granule cells of the cerebellum. We also conclusively show that the Dicer1 gene encodes a haploinsufficient tumor suppressor gene for Ptch1-induced medulloblastoma, with the monoallielic loss of Dicer1 more severe than biallelic loss. These findings exemplify how genetic interplay between pathways may produce nonadditive effects with a substantial and unpredictable impact on biology. Furthermore, these findings suggest that the functional dosage of Dicer1 may nonadditively influence a wide range of Shh-Ptch-dependent pathologies. KEYWORDS Dicer1 protein; mouse; genes; tumor suppressor; Hedgehog proteins; heterozygote; medulloblastoma; nervous system malformations

icroRNAs (miRNAs) are a class of 22-nucleotide noncoding RNA molecules that function as sequencespecific guides. They direct an Argonaute (Ago) proteincontaining RNA-induced silencing complex (RISC) to target messenger RNAs (mRNAs) (Hutvagner and Zamore 2002; Mourelatos et al. 2002) by way of Watson–Crick pairing of the miRNA seed sequence to complementary elements usu-

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Copyright © 2016 by the Genetics Society of America doi: 10.1534/genetics.115.184176 Manuscript received October 27, 2015; accepted for publication January 10, 2016; published Early Online January 13, 2016. Supporting information is available online at www.genetics.org/lookup/suppl/ doi:10.1534/genetics.115.184176/-/DC1. 1Corresponding author: Otto Hirschfeld Bldg. 81, Room 619, Chancellors Place, University of Queensland, St. Lucia, Queensland 4072, Australia. E-mail: [email protected]

ally found within the 39 untranslated region of target mRNAs (Bartel 2009). This results in translational repression and/or mRNA destabilization and decay (see Iwakawa and Tomari 2015 for a recent review). MicroRNAs are first transcribed as long primary transcripts (pri-miRNAs) with a characteristic hairpin-like secondary structure (Mourelatos et al. 2002). In the canonical bioprocessing pathway, this hairpin-like structure is recognized by the microprocessor–enzyme complex, through Ribonuclease 3 (protein Drosha) and Microprocessor complex subunit DGCR8 (Lee et al. 2003; Denli et al. 2004; Han et al. 2004), which cleaves the pri-miRNA into an 70-nucleotide precursor miRNA (pre-miRNA). The pre-miRNA is further processed, once exported to the cytoplasm (Bohnsack et al. 2004; Lund et al. 2004), by the RNase III enzyme Dicer, into a mature 22-nucleotide RNA duplex

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with 2-nucleotide overhangs at both 39 hydroxyl termini (Blaszczyk et al. 2001; Hutvagner et al. 2001). The endoribonuclease Dicer, in addition to cleaving pre-miRNAs, participates in the loading of miRNAs into RISC (Chendrimada et al. 2005). Both the endoribonuclease Dicer and protein Drosha are thought to cleave double-stranded RNAs by forming an intramolecular pseudodimer between two tandem RNase III domains (a/b) (Han et al. 2004; Zhang et al. 2004). In mice, a lysine residue (K1790) within the ribonuclease (RNase) IIIb domain of endoribonuclease Dicer appears critical for double-stranded RNA cleavage (Du et al. 2008). MicroRNAs, and by extension endoribonuclease Dicer function, are implicated in virtually every biological process in a wide range of organisms, and changes in their expression are associated with a plethora of human pathologies. In the cerebellum, miRNA dysfunction, via ablation of endoribonuclease Dicer function, has been linked to movement disorders (Lee et al. 2008; Constantin and Wainwright 2015), neurodegeneration (Schaefer et al. 2007), and the malignant neoplasm, medulloblastoma (Zindy et al. 2015). MicroRNAs play a prominent role in medulloblastoma, given that distinct miRNA expression signatures are able to classify the molecular (Cho et al. 2011) and histopathological (Ferretti et al. 2009) subtypes of human medulloblastoma, and dozens of independent studies have identified the functional networks by which specific miRNAs contribute to the growth and progression of this disease. For example, the miR-1792 cluster causally contributes to medulloblastoma and granule cell precursor proliferation through Sonic Hedgehog (Shh)Patched (Ptch) signaling (Northcott et al. 2009; Uziel et al. 2009). Similarly, miR-106b has been shown to promote the proliferation, migration, and invasion potential of medulloblastoma (Li et al. 2015), presumably via the principal mediator of the transcriptional response of the Shh-Ptch-signaling pathway, GLI-Kruppel family member GLI2 (Gli2) (Constantin and Wainwright 2015). The cerebellum is located anteriorly on the brainstem, close to the midbrain–hindbrain boundary. It is essential for the fine motor control of movement and posture and, on the basis of human lesion and functional neuroimaging studies, appears to contribute to a broad range of high-order nonmotor functions (see Buckner 2013 for review). A principal cell type of the cerebellum, and the most numerous in the central nervous system, is the granule cell. Granule cells are of critical importance to cerebellar function, as exemplified by the severity of motor coordination, language, and cognitive deficits observed in patients with congenital granule cell degeneration (Pascual-Castroviejo et al. 1994). Precursors of granule cells arise from the rhombic lip, a thin strip of neuroepithelium that borders the non-neuronal roof plate of the fourth ventricle (Alder et al. 1996). During embryogenesis, granule cell precursors leave the rhombic lip and migrate anteriorly over the outer surface of the cerebellum to form a second germinal structure, termed the “external germinal layer” (see Hatten and Roussel 2011 for review). After birth, granule cell precursors undergo rapid proliferation in the

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external germinal layer. Proliferation is maintained until perinatal life by way of multiple symmetric mitoses in singlefated germinal cells (Espinosa and Luo 2008). Once granule cells become postmitotic, they extend parallel fiber axons that synapse to the dendrites of GABAergic inhibitory Purkinje neurons, while the soma of the granule cells migrate interiorly to their final destination in the internal granular layer (Komuro and Rakic 1998). Multiple mitogenic pathways are known to drive the rapid expansion of granule cell precursors in the external germinal layer, the most potent of which is Shh (Dahmane and Ruiz i Altaba 1999; Lewis et al. 2004). In the central nervous system, the binding of Shh to the transmembrane protein Ptch relieves the inhibitory action of Ptch on the transmembrane protein Smoothened (Johnson et al. 2000; Briscoe et al. 2001). Active-state Smoothened is then able to translocate to the primary cilium (Corbit et al. 2005), where it promotes the formation of the active Gli family of transcription factors, which in turn induces the expression of target genes such as Gli1, Ptch1, and Nmyc (Humke et al. 2010; Tukachinsky et al. 2010). Therefore, the removal of Ptch from the plasma membrane, or the binding of Ptch to Shh, activates Shh-Ptch signaling. Although some miRNAs are known to interact with Hedgehog-Ptch signaling, the magnitude and directionality of this interaction is not well understood, even within the cerebellum. In the present study, we investigated the gene dose-dependent interaction between miRNA biogenesis and Shh-Ptch signaling in cerebellar granule cell precursors. The inactivation of Dicer1 in granule cell precursors is hypoplastic (Constantin and Wainwright 2015; Zindy et al. 2015). Alternatively, constitutive activation of Shh-Ptch signaling in granule cell precursors is well known to produce hyperplasia that resembles human medulloblastoma (Goodrich et al. 1997; Pietsch et al. 1997; Raffel et al. 1997). In fact, the most widely used animal model for medulloblastoma is the targeted mutation of the Ptch1 gene, either heterozygous null (Goodrich et al. 1997; Hahn et al. 1998; Zibat et al. 2009) or conditional knockouts associated with the granule neuron lineage (Ellis et al. 2003; Yang et al. 2008). Given that the two biological processes of hypoplasia and hyperplasia are on opposing growth spectrums, we hypothesized that the co-deregulation of miRNA biogenesis and Shh-Ptch-signaling pathways would attenuate overall growth and lead to reduced severity of murine medulloblastoma. To inactivate miRNA biogenesis, we used the conditional Dicer1 allele (Dicer1flox) with the knock-in mouse model, Atonal homolog 1-Cre recombinase (Atoh1-Cre, also known as Math1-Cre) to confer specificity to the granule cell lineage. Similarly, we constitutively activated Shh-Ptch signaling through the conditional Ptch1 allele (Ptch1flox), together with Atoh1-Cre. Indeed, we established that a strong genetic interaction exists between miRNA biogenesis and Shh-Ptch signaling; however, this interaction did not bring the growth phenotypes toward equilibrium. Instead, combined deregulation exasperated hypoplasia in a subset of animals and exasperated medulloblastoma in the remaining subset. These findings are of interest because they

suggest that the miRNA biogenesis and Shh-Ptch-signaling pathways positively cooperate with one another to combinatorially regulate an important developmental transition in granule cell development.

Materials and Methods Mice

Dicer1flox, Ptch1flox, and Gt(ROSA)26Sor;lacZ mice have previously been described (Soriano 1999; Ellis et al. 2003; Harfe et al. 2005). Atoh1-Cre mice (line 8) were generated in the Rowitch laboratory at the Institute for Regeneration Medicine (San Francisco) and are modified from Atoh1-Cre mice previously described (Schuller et al. 2007). Atoh1-Cre and Gt(ROSA)26Sor;lacZ mice were interbred to test the recombination efficiency and expression pattern of the Atoh1-Cre transgene. Dicer1flox/wt;Ptch1flox/flox and Atoh1-Cre;Dicer1flox/wt;Ptch1flox/wt mice were interbred to generate the experimental cohort with littermate controls. Experimental cohorts were monitored daily for signs of disease. Adult and neonatal mice with a severe disease score in any one of the following phenotypes—head swelling, respiratory changes, weight loss, unresponsiveness, poor grooming, loss of balance and/or correct posture, or abnormal movements—were euthanatized by decapitation, and immediately tissues of interest were harvested. All procedures involving animals were approved by the University of Queensland Animal Ethics Committee and were performed in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Tissue processing

Mouse brains were dissected in ice-cold phosphate-buffered saline using a stereomicroscope. One-half of the brain, in the midsagittal plane, was immersed in 4% paraformaldehyde for 12–16 hr at 4° and then processed and embedded in paraffin. From the remaining half of the brain, a 2- to 3-mm2 biopsy of the superficial-medial tumor mass or cerebellum was removed for protein harvest; the remaining tumor mass or cerebellum was stored in RNAlater reagent (Qiagen) for downstream DNA and RNA analyses. For b-galactosidase staining, embryonic tissues derived from Atoh1-Cre;Gt(ROSA)26Sor;lacZ mice were gently fixed in 2% formaldehyde and 0.2% glutaraldehyde for 2.5 hr at 4° with agitation. Following fixation, tissues were washed in phosphate-buffered saline, incubated with agitation in 30% sucrose in phosphate-buffered saline overnight at 4–10°, and then embedded in Tissue-Tek O.C.T. compound (Sakura). To extract genomic DNA, a 2- to 3-mm2 biopsy was removed from RNAlater-preserved tumors or cerebellums. Biopsies were homogenized with a sterile razor, digested with 500 mg/ml of proteinase K in 60 mM Tris, 100 mM ethylenediaminetetraacetic acid, and 0.5% sodium dodecyl sulfate at 55° for 4 hr and then were purified using a standard phenol/chloroform method. The miRNeasy mini kit (Qiagen) was used to extract total RNA from a 2- to 3-mm2 biopsy of RNAlater-preserved tissue.

Polymerase chain reaction analyses

All polymerase chain reaction (PCR) products were amplified using standard touchdown cycling conditions with a final annealing temperature of 55° and Taq DNA polymerase (Life Technologies). Cre-mediated recombination of genomic Dicer1flox was measured using the following primers: forward (59-CCT GAC AGT GAC GGT CCA AAG-39) and reverse (59-CCT GAG TAA GGC AAG TCA TTC-39). The wild-type allele produced an 1200-nt PCR product, the undisrupted loxP allele produced an 1400-nt PCR product, and the recombined gene produced an 350-nt PCR product. LoxP recombination of genomic Ptch1 was measured using the following primers: forward (59-TGG TTG TGG GTC TCC TCA TAT T-39) and reverse (59-TGA CTG CAA ACT TTC CCA TCT-39). The undisrupted gene was too large to amplify (and was therefore undetectable), while the recombined gene produced a 499-nt PCR product. LoxP recombination of Dicer1 complementary DNA (cDNA) was measured using the following primers: forward (59-AGA CGC AGA GAA AAC CCT CA-39) and reverse (59-TCT CCG CTG GGC TAA ACT T-39). The undisrupted gene produced a 656-nt PCR product while the recombined gene produced a 387-nt PCR product. Alternatively, loxP recombination of Ptch1 cDNA was measured using the following primers: forward (59-TGG TTG TGG GTC TCC TCA TAT T-39) and reverse (59-CAC CGT AAA GGA GGC TTA CCT A-39). The undisrupted gene produced a 463-nt product while the recombined gene produced a 273-nt PCR product. Complementary DNA was synthesized from 10 ng/ml of total RNA primed with Oligo(dT) (Life Technologies) by way of the SuperScript III system (Life Technologies) following the manufacturer’s instructions. MicroRNA cDNA was synthesized using a TaqMan miRNA reverse transcription kit (Applied Biosystems). Each RNA sample (5 ng/ml) was reverse-transcribed in quadruplicate with specific miRNA stem-loop primers (Applied Biosystems), including the control gene RNA, U6 small nuclear 6, pseudogene (Rnu6-2). Quantitative reverse transcriptase PCR was performed using the TaqMan universal PCR master mix with no AmpErase UNG (Applied Biosystems), in technical triplicate, following the manufacturer’s instructions. An ABI Prism 7000 Sequence Detection system with SDS version 1.2.3 software was used to detect and analyze relative expression levels. Analysis was performed using the relative threshold cycle (CT) method. Histology and imaging

Whole-mount dorsal views of the mouse cerebellum were captured during dissection using the Olympus SZX12 stereomicroscope. Gross histological analyses were performed using standard (Luna et al. 1968) hematoxylin and eosin Y (H&E) staining for 3.5 min and 45 sec, respectively, on 7-mm-thick midsagittal sections of paraffin-preserved mouse brains. Paired box protein Pax-6 (Pax6) and neuronal nuclei (NeuN) co-immunolabeling was performed sequentially. Specifically, 7-mm-thick midsagittal sections of paraffin-preserved mouse

Pathway Interactions in the Cerebellum

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brains were boiled for 3 min in citric acid-based Antigen Unmasking Solution (Vector Laboratories). Sections were blocked in 10% animal serum, 2% bovine serum albumin, and 0.025% Triton X-100 in phosphate-buffered saline for 1 hr and then incubated overnight at 4–10° with the first primary antibody, Pax6 (1:200, Covance), and then with secondary antibody conjugated to Alexa Fluro 555 (1:250, Invitrogen) for 1 hr at room temperature. Sections were again blocked in 10% animal serum, 2% bovine serum albumin, and 0.025% Triton X-100 in phosphate-buffered saline for 1 hr at room temperature and incubated for 2 hr at room temperature with the second primary antibody NeuN (1:200, Merck/Millipore) and then with secondary antibody conjugated to Alexa 488 (1:250, Invitrogen) for 1 hr at room temperature. Sections were counterstained with 0.1 mg/ml of 49,6-diamidino-2-phenylindole, mounted in Fluorescent Mounting (Dako) medium, and visualized using the LSM 510 Meta UV (Zeiss) confocal microscope. For b-galactosidase (lacZ) staining, 7-mm-thick midsagittal cryopreserved sections were incubated in 0.2% gluteraldehyde, 50 mM ethylene glycol tetraacetic acid, and 100 mM magnesium chloride in phosphate-buffered saline for 10 min at 4° and were then washed in lacZ wash (2 mM magnesium chloride, 0.1% sodium deoxycholate, 0.02% NP40 in phosphatebuffered saline). Subsequently, sections were incubated at room temperature with 0.5 mg/ml X-gal, 5 mM potassium ferracynaide, and 5 mM potassium ferricynaide in lacZ wash until color development. Sections were counterstained with Nuclear Fast Red (Sigma-Aldrich) for 4 min. Western blot

Directly after dissection, tissue biopsies were homogenized with a 27G 1/2-inch needle in 20 mM Tris–hydrochloride (pH 7.5) with protease inhibitors. Protein concentrations were estimated using the BCA (Pierce) protein assay kit. Five micrograms of denatured protein were separated on a 4.5% resolving gel by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were then transferred to a 0.45-mm Immobilon-P (Merck Millipore) polyvinylidene fluoride membrane by electroblotting at 30 mA for 16 hr at 4°. Membranes were blocked in 5% skim milk in TBS-Tween for 1 hr, incubated with the primary antibodies Dicer 349 (1:5000; kind gift from Witold Filipowicz) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:7000, Trevigen), and then incubated with IgG and HRP-conjugated antibodies (1:5000, Sigma) for 2 hr at room temperature. Proteins were visualized with ECL Plus (GE Healthcare) Western blotting detection reagents and imaged with the GS-800 Calibrated Densitometer (Bio-Rad). Theoretical molecular weights were calculated using the ExPASy server and known amino acid sequence. In situ hybridization

Fourteen-micrometer-thick RNase-free sagittal sections of the central nervous system, medial to the vermis, were labeled using 500 ng of the Atoh1 riboprobe as previously described

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(Adolphe et al. 2004). The Atoh1 riboprobe was generated using the primers SP6 forward (59-GCG ATT TAG GTG ACA CTA TAG GAC CAC CAT CAC CTT CGC-39) and T7 reverse (59-GCG TGT ATT CTG GGT CCG CCC TAT AGT GAG TCG TAT TAA CAT CG-39) to create a 877-nt product. DICER1 expression vector

Inverse PCR was performed on pBluescript II SK(+)-DICER1 (Zhang et al. 2002) with KOD hot start DNA polymerase (Merck) and primers oriented in the reverse direction of, yet flanking, exons 24 and 25 of human DICER1 to amplify the entire construct but without exons 24 and 25 (Dex24-25). Primers used were exon 26 forward [59(P)-AAA AGT TTT CTG CAA ATG TACC-39] and exon 23 reverse (59-CAG TGA TAG TAT TGT AGT GG-39). Modifications to the manufacturer’s guidelines were the addition of 1.5 mM of magnesium sulfate, an annealing temperature of 52°, and an extension of 5 m and 30 sec over 20 cycles. DICER1Dex24-25 vs. DICER1 wild-type products were screened by XbaI and NotI restriction enzyme digest to identify 3229- vs. 3661-nt DNA products, respectively, and then sequencing of the described fragment was performed. To reduce the number of potential mutations outside of the region sequenced, a confirmed XbaIand NotI-digested DICER1Dex24-25 fragment was subcloned into the original pBluescript II SK(+)-DICER1 construct. To measure transfection efficiency with an enhanced GFP (eGFP), wild-type and mutant DICER1 were excised from pBluescript II SK(+) using the restriction enzyme KpnI and NotI, blunted using T4 DNA polymerase (New England Biolabs), and then ligated into an EcoRV restriction site of the pCIG2-IRES-eGFP vector (Grainger et al. 2010). Tissue culture

Control and Dicer1 null sarcoma (Ravi et al. 2012) and mesenchymal stem [American Type Culture Collection (ATCC) CRL-3221] cells were kindly donated by the Phillip Sharp laboratory (Massachusetts Institute of Technology). These cells were maintained in culture as previously described (Ravi et al. 2012; Gurtan et al. 2013). COS-7 cells (ATCC CRL-1651) were maintained as described by the ATCC. For transfection experiments, cell lines were seeded in a 24-well plate and transfected at 70% confluence with Lipofectamine LTX and Plus reagents (Life Technologies) and 500 ng of pCIG2IRES-eGFP, wild-type pCIG2-DICER1-IRES-eGFP, or mutant pCIG2-DICER1Dex24-25-IRES-eGFP. Total RNA was harvested from COS-7 and sarcoma cells 24 hr after transfection and 36 hr following transfection in mesenchymal stem cells. Statistics

Tumor-free survival of Atoh1-Cre; Ptch1flox/flox; Dicer1flox/ wt and Atoh1-Cre; Ptch1flox/flox; Dicer1flox/flox, when compared to Atoh1-Cre; Ptch1flox/flox mice, was statistically significant according to both the log-rank (Mantel–Cox) and Gehan–Breslow–Wilcoxon tests. Deaths not resulting from medulloblastoma were censored in Kaplan–Meier survival estimates (see Supporting Information, Table S1).

Figure 1 Genomic Ptch1flox and Dicer1flox are inactivated in medulloblastoma. Semiquantitative PCR analyses of genomic DNA extracted from Atoh1-Cre;Ptch1flox tumors confirmed the efficient inactivation of Ptch1flox (A) and Dicer1flox (C) by Cre-mediated recombination. Specifically, Ptch1 recombination produced an 500nucleotide (nt) product, while the loxP and wild-type alleles, respectively, produced 350- and 300-nt products (A). Furthermore, Dicer1 inactivation of the loxP allele (1400 nt) produced an 350-nt product, while the wild-type allele generated an 1200-nt product (C). The location of primer binding (F, forward primer; R, reverse primer) relative to exons (numerical) is depicted schematically underneath representative images of DNA agarose gel electrophoresis (A and C). Atoh1-Cre is expressed uniformly in the external germinal layer (solid arrowhead) from embryonic day 18.5 (B), as demonstrated by b-galactosidase staining (blue precipitate) in Atoh1-Cre;Gt(ROSA)26Sor;lacZ mice. Sections were counterstained with nuclear fast red. Bars in B: top, 1 mm; bottom, 50 mm.

Results Dicer1 function was efficiently disrupted in Atoh1-Cre; Ptch1flox/flox mice

To investigate the dose-dependent interactions between miRNA biogenesis and Shh-Ptch signaling in cerebellar granule cells, Atoh1-Cre mice were interbred with mice carrying the Dicer1flox and Ptch1flox alleles. The most widely used animal model for medulloblastoma involves the targeted mutation of the Ptch1 gene, either heterozygous null (Goodrich et al. 1997; Hahn et al. 1998; Zibat et al. 2009) or conditional knockouts associated with the granule cell lineage (Yang et al. 2008; Li et al. 2013), which leads to the constitutive activation of Shh-Ptch signaling in the cerebellum. In these mouse models, including Ptch1 heterozygous null mutants, the absence of Ptch1 mRNA (and ensuing Shh-Ptch-pathway activation) is critical for both the initiation and growth of medulloblastoma. For example, Hedgehog pathway inhibitors cause tumor regression in mouse models of medulloblastoma (Yauch et al. 2009; Buonamici et al. 2010; Lee et al. 2012; Rohner et al. 2012), and the emergence of resistance to these inhibitors is always liked to downstream activation of Shh-Ptch signaling (Yauch et al. 2009; Buonamici et al. 2010; Dijkgraaf et al. 2011). Although the Atoh1-Cre;Ptch1flox/flox mouse model used in the present study has previously been characterized (Yang et al. 2008), Ptch1 recombination in the presence of the Dicer1flox allele was measured by semiquantitative PCR on genomic DNA isolated from Ptch1flox and Atoh1-Cre;Dicer1flox/flox tumors. Indeed, Ptch1 recombination was detected (by the presence of a 499-nt PCR product) in tumors with homozygous Dicer1 inactivation (Figure 1A). Unlike the Ptch1 mouse model, Atoh1-Cre;Dicer1flox/flox mice are not well characterized in the literature. Briefly, the Dicer1flox mouse possesses loxP sites that flank exon 24 of the Dicer1 gene (Harfe et al. 2005). Exon 24 encodes the Nterminal quarter of the RNase IIIb domain and a critical lysine residue (K1790) required for the 59 phosphate cleavage of pre-miRNA duplexes (Du et al. 2008). The removal of exon

24 is predicted to inactivate endoribonuclease Dicer function by producing a nonsense transcript with a premature stop codon in the second amino acid after the recombination site. Other widely used Dicer1flox mouse models possess loxP sites flanking exons 22–23 (Murchison et al. 2005) and exons 15–17 (Mudhasani et al. 2008). To better characterize Atoh1-Cre; Dicer1flox/flox mice, the recombination efficiency and expression pattern of Cre recombinase under the Atoh1 promoter was investigated by crossing Atoh1-Cre transgenic mice with mice carrying the Gt(ROSA)26Sor;lacZ allele. Homozygous Gt(ROSA)26Sor;lacZ mice contain a loxP-flanked DNA stop sequence that prevents the downstream expression of lacZ (Soriano 1999). When these mice are crossed with a Cre transgenic line, the DNA stop sequence is removed such that lacZ is expressed in the Cre-positive tissues only. Beta-galactosidase staining of Atoh1-Cre;Gt(ROSA)26Sor;lacZ mice confirmed the efficient Cre-mediated recombination in the external germinal layer of the cerebellum from embryonic day 18.5 (Figure 1B). Next, the level of Dicer1 recombination was investigated by semiquantitative PCR on genomic DNA derived from Atoh1-Cre; Ptch1flox/flox tumors. Indeed, a high level of Dicer1 recombination was detected in tumors isolated from Atoh1-Cre;Ptch1flox mice (Figure 1C) by comparing the relative intensity of the Dicer1 deleted band (350 nt) to the Dicer1flox band (1400 nt) (Figure 1C). Together, these data verify that genomic Dicer1 and Ptch1 are efficiently recombined in medulloblastoma. The expression levels of recombined Ptch1 mRNA (Figure 2, A and B) and Dicer1 mRNA (Figure 2, C and D) were next assessed by reverse transcriptase PCR. Specifically, recombination of Ptch1 transcripts were classified as incomplete (open), partial (shaded), or near-complete (solid) based on the relative levels of Ptch1 reverse-transcribed PCR products missing loxP-flanked exon 3 (273 nt) compared to PCR products with exon 3 (463 nt) (Figure 2, A and B). Near-complete Ptch1 recombination was detected in medulloblastoma, regardless of whether the tumors originated from a Atoh1-Cre; Ptch1flox/wt or Atoh1-Cre;Ptch1flox/flox tumor or the status of Dicer1 (Figure 2E). This is consistent with previous reports


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Figure 2 Ptch1flox and Dicer1flox transcripts are inactivated in medulloblastoma and hypoplastic cerebellums. Semiquantitative PCR analyses of reverse-transcribed RNA extracted from the cerebellums of mice, prior to medulloblastoma or hypoplasia-induced death, were examined for the comparative levels of Ptch11flox (A and B) and Dicer1flox (B–D) Cre-mediated recombination. Schematic diagrams (A and C) depict the variety of splice and recombined transcripts observed in cerebella, with the predicated size of PCR products in nucleotides (nt). Cre-meditated recombination of Ptch1 (Ptch1Dex3) produced a 273-nt product (C and D). Cre-meditated recombination of Dicer1 (Dicer1Dex24) produced a 387- or 224-nt product (A and B). The 224-nt product was produced because of an endogenously expressed, alternatively spliced exon 25 variant (Dicer1Dex25; 387 nt) that also underwent recombination (Dicer1Dex24-25). The comparative levels of recombination for Ptch1 and Dicer1 mRNA were classified as the following: incomplete (open box), partial (shaded box), or near-complete (solid box) recombination, as shown in the representative images of DNA agarose gel electrophoresis (B and D). Information was tabulated according to genotype, cause of death, and days alive (E). H, hypoplasia; Hh, hypoplasia with hydrocephalus; MB, medulloblastoma. Schematic diagrams (A and C) are drawn to scale.

(Berman et al. 2002) that silencing of wild-type Ptch1 is necessary for the initiation of Shh-Ptch signaling and medulloblastoma growth in Ptch1 mouse models. On the other hand, there was an incomplete or partial loss of Ptch1 in Atoh1-Cre; Ptch1flox/wt;Dicer1flox/flox mice that developed hypoplasia (Figure 2E). Similarly, recombination of Dicer1 transcripts were classified as incomplete (open), partial (shaded) or near-complete (solid) based on the relative levels of Dicer1 reverse-transcribed PCR products missing loxP-flanked exon 24 (387 and 224 nt) compared to PCR products with exon 24 (656 and 493 nt) (Figure 2, C and D). In Atoh1-Cre;Ptch1flox/ wt;Dicer1flox/flox and Atoh1-Cre;Ptch1flox/flox;Dicer1flox/ flox mice, Dicer1 in most cases was completely, or partially, recombined, regardless of the cause of death (by medulloblastoma or hypoplasia) (Figure 2E). However, Atoh1-Cre; Ptch1flox/flox;Dicer1flox/wt mice were incompletely or partially recombined (Figure 2E). These data suggest that Ptch1 mRNA is not under the same selective pressure for loss of expression in hypoplastic cerebella as it is in medulloblastoma, and that wild-type Dicer1 mRNA is not under selective pressure for loss in Atoh1-Cre;Dicer1flox/wt tumors with Ptch1 loss. Endogenous Dicer1 splice variants do not affect microRNA bioprocessing

An endogenously expressed exon 25 splice variant was identified in medulloblastoma and hypoplastic cerebella based on

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amplicon size (494 nt vs. wild type of 657 nt) (Figure 2, C and D) and sequencing (data not shown). Interestingly, the exon 25 splice variant underwent simultaneous Cre-meditated recombination to produce an exon 24–25 deletion (225-nt PCR product) (Figure 2, C and D). Furthermore, Cre-mediated recombination appeared to promote exon 25 skipping, as evidenced by a relatively higher proportion of Dicer1 exon 24–25 deleted transcripts compared to Dicer1 exon 25 alone being deleted (Figure 2D). Dicer1 has previously been demonstrated to undergo exon 25 alternative splicing in human primary neuroblastic tumors that originate from neural crest tissues (Potenza et al. 2010). A potential mechanism to promote exon skipping may involve the presence of somatic single-base substitutions in the RNase IIIb domain that introduce a splicing silencer (Wu et al. 2013). The excision of exons 24 and 25 is predicted to keep the transcript inframe, but remove the majority (80%) of the RNase IIIb domain. Alternative splicing of exon 25 would affect the Dicer1 flox mouse model used in the present study (Harfe et al. 2005), but not the other widely used Dicer1flox mouse models by Murchison et al. (2005) and Mudhasani et al. (2008), given that their loxP sites do not immediately flank exon 25. To determine whether Dicer1 lacking exons 24–25 was functional, sarcoma and mesenchymal stem cells deficient in Dicer1 were transfected with full-length DICER1 or DICER1 lacking exons 24–25 (DICER1Dex24-25). Sarcoma and

Figure 3 Excision of exon 24–25 of DICER1 inactivated microRNA bioprocessing. Quantitative PCR of reverse-transcribed RNA verified that the protein products of the DICER1Dex24 (medium shading) and DICER1Dex24-25 (light shading) were unable to bioprocess three highly expressed microRNAs in Dicer1 null sarcoma cells (A) and Dicer1 null mesenchymal stem cells (B), while the protein product from DICER1 (solid bar) was able to. Quantitative realtime PCR analyses were calculated using the relative threshold cycle (CT) method (A and B) relative to the housekeeping gene Rnu6-2. Stable expression of the endoribonuclease Dicer splice and recombination variants was confirmed by Western blot analyses of COS-7 cells transfected with various DICER1 constructs (C). Furthermore, Western blot analysis of endogenous endoribonuclease Dicer protein levels was performed on tumors derived from Atoh1Cre;Ptch1flox/flox mice (D). Protein levels were substantially reduced in tumors derived from Atoh1-Cre;Ptch1flox/flox;Dicer1flox/flox mice. GAPDH was used as a housekeeping protein (D). Representative Western blot images are shown (C and D).

mesenchymal stem cells were specifically chosen because they are able to tolerate null mutations in Dicer1, while most other cell types cannot, as exemplified by the requirement for Dicer1 in early mouse development (Bernstein et al. 2003). Exon 24–25 exclusion did not alter basal miRNA biogenesis in either of the Dicer1 null cell lines investigated, as illustrated by the comparable relative expression level of miRNAs between empty vector and DICER1Dex24-25 transfected cells (Figure 3, A and B). These data demonstrate that exon 24–25 exclusion produces a loss-of-function mutation equivalent to the recombination of Dicer1flox (i.e., exon 24 excision) alone. To further validate that the various DICER1 constructs lacking exons 24 and/or 25 produced stable protein products, Western blot analyses were performed. Indeed, all constructs produced protein products of the expected size that was based on the predicted amino acid sequence of the endoribonuclease Dicer: 217 kDa for DICER1 (estimated 216 kDa), 192 kDa for DICER1Dex24 (estimated 206 kDa), 207 kDa for DICER1Dex25 (estimated 210 kDa), and 212 kDa for DICER1Dex24-25 (estimated 200 kDa) (Figure 3C). Interestingly, in primary Atoh1-Cre;Ptch1flox/flox tumors, regardless of the status of Dicer1flox, splice variants for exon 24 and/or 25 were not detected (Figure 3D), even though they were detectable at the transcript level (Figure 2, C–E). This does not preclude the possibility that low levels of alternatively spliced endoribonuclease Dicer would be able to influence tumor growth; however, given that DICER1Dex24-25 is functionally equivalent to Dicer1flox (i.e., exon 24 excision), low undetectable levels of endoribonuclease Dicer lacking exons

24–25 would still produce a loss-of-function mutation. Therefore, these data collectively demonstrate that endoribonuclease Dicer function, regardless of exon 25 skipping, is efficiently disrupted in Ptch1-induced cerebellar tumors. Dicer1 positively corroborates with Sonic HedgehogPatched signaling in granule cell precursors

To understand how loss of Dicer1 function influences ShhPtch signaling, the allelic frequency of Dicer1 was incrementally altered within granule cell precursors of Atoh1-Cre; Ptch1flox/flox mice. Given that homozygous and heterozygous deletion of Ptch1 (to activate Shh-Ptch signaling in granule cell precursors) induces hyperplasia that histologically resembles human medulloblastoma (Yang et al. 2008) and, conversely, that the homozygous deletion of Dicer1 in granule cell precursors produces mild hypoplasia (Constantin and Wainwright 2015) (while heterozygous deletion has no effect), these two opposing growth phenotypes of hyperplasia and hypoplasia were used as a measure of pathway activity for Shh-Ptch signaling and endoribonuclease Dicer biogenesis pathways, respectively. The severity of medulloblastoma was measured as a function of tumor-free survival (Figure 4A) with the heterozygous inactivation of Dicer1 most severe, followed by homozygous inactivation. More specifically, monoallelic inactivation of Dicer1 reduced tumor-free survival to a median of 25 days (Figure 4, B and E), while homozygous inactivation reduced tumor-free survival to a median of 62 days (Figure 4, B and F), compared to control Atoh1-Cre; Ptch1flox/flox mice, which developed medulloblastoma in a


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Figure 4 Dicer1 inactivation reduced tumor-free survival or induced hypoplasia in Atoh1-Cre;Ptch1flox/flox mice. The percentage of frequencies and severity of medulloblastoma (positive scale) and hypoplasia (negative scale) was substantially altered by the allelic dose of Dicer1 (A) inactivation in Atoh1-Cre;Ptch1flox/flox mice. Relative disease severity was ranked from least (light shading) to most severe (solid) for medulloblastoma and, similarly for hypoplasia, for the genotypes graphed only. Tumor-free percentage of survival (B) was reduced by the heterozygous (shaded solid line) and homozygous (shaded dashed line) inactivation of Dicer1 in Atoh1-Cre;Ptch1flox/flox mice. Deaths not caused by tumor load were censored (solid vertical line). Tumor-free survival in Dicer1flox heterozygous and homozygous Atoh1-Cre;Ptch1flox/flox mice was statistically significant compared to Atoh1-Cre;Ptch1flox/flox mice (****P # 0.0001). For each of the disease phenotypes depicted in the graph (A), the following representative images (C– G) were captured: whole-mount analysis of the cerebellum from the ventral-posterior aspect (left top), H&E staining (right top), immunofluorescence staining for neuronal differentiation by Pax6 and NeuN costaining (left bottom), and in situ hybridization of Atoh1 (right bottom). Some genotypes developed one of the two opposing phenotypes: hyperplasia leading to medulloblastoma (E) or hypoplasia leading to a severely underdeveloped cerebellum (F). Dashed white line marks the cerebellar boundary, and an asterisk denotes hydrocephalus. Bars in C–G: top left, 2 mm; top right, 500 mm; bottom, 200 mm.

median of 145.5 days (Figure 4, B and D). These data demonstrate that Dicer1 loss, whether monoallelic or biallelic, substantially and significantly promoted tumor progression in granule cell precursors by collaborating with Shh-Ptch signaling. Therefore, Dicer1 functions as a tumor suppressor gene, given that loss of expression enhanced tumor severity. Further to this, monoallelic Dicer1 loss exerted an effect, which indicates that Dicer1 is also a haploinsufficient tumor suppressor gene. These findings are consistent with the role of Dicer1 in other tumor types (Arrate et al. 2010; Lambertz et al. 2010; Nittner et al. 2012; Yoshikawa et al. 2013; Zhang et al. 2014). The severity of hypoplasia was calculated based on the size and fissure formation of the cerebellum at the time of death. Approximately 39% (n = 7/18) of Atoh1-Cre;Ptch1flox/flox mice with a homozygous loss of Dicer1 developed severe

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hypoplasia with hydrocephalus (Figure 4, A and G). More specifically, cerebella were of an embryonic appearance based on fissure formation (Figure 4G) and size (1.69 mm 6 0.030; mean 6 SEM; n = 4), and mice died at a median of 14 days (Table S1). The presence of the completing growth phenotype of hypoplasia in biallelically inactivated (but not in monoallelically inactivated) Dicer1 mice could explain why haploinsufficiency was more severe than near complete loss of function of Dicer1. More specifically, hypoplasia involves cellular growth processes that are in direct opposition to hyperplasia; therefore, the coexistence of these two phenotypes (such as observed in biallelically inactivated Dicer1 mice) would subdue the severity of both phenotypes compared to monoallelically inactivated Dicer1 mice that present with medulloblastoma only.

In addition to gross histological analyses of these phenotypes through H&E staining, cerebella were costained with antibodies against the granule cell lineage marker Pax6 and the neuronal differentiation marker NeuN. By identifying Pax6-immunopositive and NeuN-immunonegative cells vs. Pax6-immunopositive and NeuN-immunopositive cells, the populations of undifferentiated granule cell precursors vs. differentiated granule cells could, respectively, be determined. Pax6 and NeuN colocalization has previously been used to examine changes in the population of proliferating and differentiating granule cell precursors (Flora et al. 2009; Constantin and Wainwright 2015). As expected, there was a large number of Pax6-immunopositive and NeuN-immunonegative cells in all tumors (regardless of the allelic frequency of Dicer1), which demarcate precursors of the granule lineage (Figure 4, D–F). On the other hand, the hypoplastic phenotype was composed of Pax6-immunopositive and NeuN-immunopositive cells, which indicates the presence of differentiated cells in the majority of the hypoplastic structure with a thin layer of precursor cells superficially (Figure 4G). This suggests that the hypoplasia is a developmental defect rather than a small tumor mass. High Atoh1 expression is consistently and exclusively associated with the SHH subtype of medulloblastoma (Swartling et al. 2012). All tumors, regardless of the allelic frequency of Dicer1, were positive for Atoh1 (Figure 4, D–F), which indicates that these tumors likely belong to the Shh subtype. Hedgehog-Patched signaling collaborates with microRNA biogenesis in granule cell precursors

To understand how constitutive activation of Shh-Ptch signaling (via Ptch1 loss) influences the Dicer1 biogenesis pathway, the allelic frequency of Ptch1 was incrementally altered in granule cell precursors of Atoh1-Cre;Dicer1flox/flox mice. Again, the severity of medulloblastoma was measured as a function of tumor-free survival. In this case, Ptch1 inactivation promoted tumor severity and penetrance stepwise, with homozygous inactivation the most severe (Figure 5A). More specifically, control Atoh1-Cre;Dicer1flox/flox mice did not develop medulloblastoma (Figure 5, B and C), which validates previous findings that Dicer1 loss is insufficient for de novo tumor formation (Zindy et al. 2015). Instead, these mice developed anteriorly confined hypoplasia with complete penetrance (Figure 5C) that did not impact survival (Constantin and Wainwright 2015). Monoallelic Ptch1 inactivation reduced survival to a median of 58 days at 19% penetrance (n = 5/26) (Figure 5, B and D), while biallelic loss reduced survival to a median of 62 days with complete penetrance (Figure 5B and Figure 4E). These data indicate that Shh-Ptch signaling also positively collaborates with Dicer1 biogenesis (especially considering that Atoh1-Cre;Ptch1flox/flox mice develop medulloblastoma by a median of 145.5 days) (Figure 4A) and suggest that Shh-Ptch and miRNA biogenesis pathways bilaterally cross-talk in medulloblastoma. Similarly, allelic inactivation of Ptch1 to activate Shh-Ptch signaling increased the relative severity of hypoplasia, based

on the calculated developmental attenuation of the cerebellum at the time of death, and also penetrance. For example, the monoallelic deletion of Ptch1 resulted in a developmentally attenuated cerebellum that resembled that of a postnatal day 2 structure in size and foliation with 19% penetrance (Figure 5E), even though mice survived to a median of 14 days (Figure 5E). When Ptch1 was biallelically inactivated, the cerebellum resembled an embryonic structure as previously described (Figure 4F) with a penetrance of 39% (n = 7/18). Therefore, ShhPtch signaling collaborates with Dicer1 biogenesis to promote the developmental attenuation of the cerebellum. To confirm the pathologic phenotypes of hypoplasia and medulloblastoma in the present study, cerebella were sequentially costained with antibodies against Pax6 and NeuN. Again, tumors were composed of a large number of undifferentiated precursors of the granule lineage (Pax6-immunopositive; NeuN-immunonegative) (Figure 5D), while the hypoplasia contained no precursor populations but Pax6positive and NeuN-immunopositive cells or Pax6-immunonegative cell populations (Figure 5E). These data confirm that the hypoplasia is not a small tumor mass, but most likely resulted from a developmental delay. Similarly, through Atoh1 in situ hybridization, we were able to confirm that the tumors likely belong to the Shh subtype (Figure 5, D and E).

Discussion In the present study, the dose-dependent roles of miRNA biogenesis and Shh-Ptch signaling were investigated through the monoallelic and biallelic inactivation of Dicer1 and Ptch1. Consistent with previous reports, homozygous inactivation of Dicer1 in granule cells produced mild cerebellar hypoplasia (Constantin and Wainwright 2015), while conditional hemizygous and homozygous Ptch1 inactivation induced medulloblastoma (Yang et al. 2008). However, when different allelic frequencies of Dicer1 and Ptch1 were together perturbed, three new observations were made. First, only genotypes with biallelic Dicer1 inactivation and either partial or complete Ptch1 inactivation presented with hypoplasia and medulloblastoma. Second, concurrent inactivation of Dicer1 and Ptch1 led to more severe phenotypes for medulloblastoma and hypoplasia than the inactivation of either gene alone, which indicates that these pathways corroborate in Atoh1-positive granule cell precursors. Third, hypoplasia was progressively more severe with the dose-dependent inactivation of Ptch1, while monoallelic Dicer1 inactivation had a more detrimental effect than bialleic Dicer1 loss in medulloblastoma. Dicer1 haploinsufficeny in Ptch1-induced medulloblastoma may have resulted from the coexistence of the two opposing growth processes of hyperplasia and hypoplasia in tumors, which occurred in mice with biallelic Dicer1 loss. This would placate the severity of either phenotype, but not in tumors with monoallelic Dicer1 loss, given that these mice develop only medulloblastoma and therefore genetic suppression of the hyperplasia (by the opposing growth effect of hypoplasia) would not take place.


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Figure 5 Ptch1 inactivation reduced tumor-free survival or promoted hypoplasia in Atoh1-Cre;Dicer1flox/flox mice. The percentage of frequencies and severity of medulloblastoma (positive scale) and hypoplasia (negative scale) were substantially altered by the allelic dose of Ptch1 (A) inactivation in Atoh1-Cre;Dicer1flox/flox mice. Relative disease severity was ranked from least (light shading) to most severe (solid) for medulloblastoma and, similarly for hypoplasia, for the genotypes graphed only. Tumor-free percentage of survival (B) was reduced by the heterozygous (shaded line) and homozygous (shaded dashed line) inactivation of Ptch1 in Atoh1-Cre;Dicer1flox/flox mice. Deaths not caused by tumor load were censored (solid line). Tumor-free survival in Ptch1 heterozygous and homozygous Atoh1-Cre;Dicer1flox/flox mice was statistically significant compared to Atoh1-Cre;Dicer1flox/flox mice (*P = 0.0251; ****P # 0.0001). For each of the disease phenotypes depicted in the graph (A), the following representative images (C–E) were captured: whole-mount analysis of the cerebellum from the ventral-posterior aspect (left top), H&E staining (right top), immunofluorescence staining for neuronal differentiation by Pax6 and NeuN costaining (left bottom), and in situ hybridization of Atoh1 (right bottom). Some genotypes developed one of the two opposing phenotypes: hyperplasia leading to medulloblastoma (D) or hypoplasia leading to a severely underdeveloped cerebellum (E). Dashed white line marks the cerebellar boundary. Bars in C–E: top left, 2 mm; top right, 500 mm; bottom, 200 mm.

MicroRNA biogenesis and Hedgehog-Patched-signaling pathways genetically interact to accentuate either medulloblastoma or hypoplasia

Several miRNA subsets are known to regulate components of Shh-Ptch signaling in the cerebellum. For example, miR-106b positively regulates Gli2 transcription to promote granule cell expansion (Constantin and Wainwright 2015). The miR18396182 cluster synergizes with exogenous Shh ligand to promote the proliferation of Ptch1-induced medulloblastoma (Zhang et al. 2013); similarly, the miR-1792 cluster is necessary for Ptch1-induced medulloblastoma (Zindy et al.

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2014) through N-myc (Northcott et al. 2009). Conversely, miR-125b, miR-326, and miR-324-5p antagonize the pathway activators Smoothened and/or Gli1 to inhibit granule cell precursor growth (Ferretti et al. 2008). Therefore, it follows that the deletion of miR-106b, miR-18396182, and miR1792 likely induced hypoplasia in Atoh1-Cre;Ptch1flox/flox; Dicer1flox/flox mice, while the loss of miR-125b, miR-326, and miR-324-5p promoted medulloblastoma. Given that Dicer1 regulates the bioprocessing of hundreds of miRNAs, and in turn each miRNA may regulate hundreds of targets, Dicer1 ablation would also affect many other pathways in

addition to Shh-Ptch signaling. Also, Dicer1 ablation may influence Shh-Ptch signaling noncanonically, for example, by way of transforming growth factor b (Dennler et al. 2009). Although this body of work did not specify how or what the nature of the interactions between the endoribonuclease Dicer and Shh-Ptch signaling were, it did characterize the broader effects of this genetic interaction. Through doing so it established that Dicer bioprocessing and Shh-Ptch signaling, together, have a substantial and unexpected biological impact on the developing cerebellum (as measured by tumor latency and growth attenuation) and that this interaction is a principal driving event during cerebellar development. Bistability of phenotypes suggests that the associated pathways regulate an important developmental transition

A bistable phenotype was induced in Atoh1-Cre;Ptch1flox/ flox;Dicer1flox/flox mice, which strongly suggests that miRNA biogenesis and Hedgehog-Patched signaling together regulate an important developmental transition in granule cell precursors. Based on the developmental stage of cerebellar attenuation in Atoh1-Cre;Ptch1flox/flox;Dicer1flox/flox mice, such an event likely occurred in late embryonic development. Further support of the molecular switch phenomenon includes previous findings that miRNAs regulate transcription factors associated with Hedgehog-Patched signaling. More specifically, interactions between miRNAs and transcription factors permit feedback and feed-forward loops to be established, which are required in genetic switches (Tsang et al. 2007). A similar phenomenon may occur in granule cell precursors, whereby miRNA-mediate control of Shh-Ptch transcription factors, such as Gli1/2 and N-myc, allows feedback and feed-forward loops to be produced that in turn mediate hypoplasia over medulloblastoma, or vice versa. The next step would be to identify the specific miRNAs and intracellular components involved in regulating such a developmental transition. This would not only cultivate a more thorough understanding of early granule cell precursor development by, for example, shedding light on relatively unstudied molecular processes involved in establishing the developmental compartment-like units of the cerebellum (Hutvagner and Zamore 2002), but may also yield novel insights into the molecular basis of Shh-induced medulloblastoma. Dicer1 functions as a haploinsufficient tumor suppressor gene in Ptch1-induced medulloblastoma

DICER1 has previously been reported to function as a haploinsufficient tumor suppressor gene in human tumors (Karube et al. 2005; Chiosea et al. 2007; Merritt et al. 2008; Grelier et al. 2009; Faggad et al. 2010; Lin et al. 2010; Khoshnaw et al. 2012; Jafarnejad et al. 2013) and genetically engineered mouse models (Kumar et al. 2009; Lambertz et al. 2010), with only a handful of exceptions (Arrate et al. 2010; Sabbaghian et al. 2012; Zhang et al. 2013). The precise role of DICER1 in medulloblastoma is less clear. For example, subsets of miRNAs have been shown as either growth-inhibitory

(Ferretti et al. 2008; Li et al. 2009; Venkataraman et al. 2013; Jin et al. 2014; Hemmesi et al. 2015) or oncogenic (Grunder et al. 2011; Weeraratne et al. 2012; Li et al. 2015). In particular, the miR-1792 cluster has been shown as both necessary and sufficient to initiate medulloblastoma (Zindy et al. 2014), which suggests an oncogenic role for Dicer1 on the basis of miR-1792 alone. On the other hand, the global reduction of miRNAs in medulloblastoma (Ferretti et al. 2008), together with the identification of a germline DICER1 truncation mutation in a medulloblastoma cohort (Slade et al. 2011), suggests that DICER1 is tumor-suppressive and haploinsufficient. Recently, Dicer1 loss was shown to accelerate tumor growth in a genetically engineered mouse model of Ptch1induced medulloblastoma (Zindy et al. 2015); however, biallelic loss was never investigated because of the widespread apoptosis and ensuing perinatal lethality induced by Dicer1 ablation in the Murchison et al. (2005) mouse model. In the present study, we conclusively demonstrate that both the heterozygous and homozygous loss of Dicer1 dramatically decreases the latency of tumor-free survival in a genetically engineered mouse model of Ptch1-induced medulloblastoma—different from that used by Zindy et al. (2015)—and that heterozygous Dicer1 inactivation is most severe. The data presented here are consistent with the role of DICER1 in the majority of human tumors. Interestingly, to date, only a single germline mutation (of 86 cases) has been reported for DICER1 in medulloblastoma and infratentorial primitive neuroectodermal tumors (Slade et al. 2011), which suggests that the genetic interaction between miRNAs and Shh-Ptch signaling is perhaps more modest in humans or that mutations may affect this pathway downstream/post-transcriptionally to DICER1 (mRNA). Further to this, our data imply a mechanism as to why monoallelic Dicer1 inactivation is more severe than biallelic inactivation. That is, given that hypoplasia is on the opposite growth spectrum to medulloblastoma, and that only homozygous Dicer1 inactivation induces hypoplasia, the biallelic loss of Dicer1 function would genetically suppress the severity of hyperplasia, leading to medulloblastoma. This would result in the less severe tumor phenotype in mice with biallelic Dicer1 loss, as was observed. This may also explain haploinsufficiency in other developmental pathologies. Tumor suppressor genes can be designated as gatekeeper genes, for those involved in the control of cell proliferation or cell death, or as caretaker genes, for those involved in the control of DNA integrity, as reviewed by Kinzler and Vogelstein (1997). The inactivation of a caretaker gene often does not initiate tumor formation, but fosters transformation by rendering the cell genetically unstable. The endoribonuclease Dicer shares similarities with the majority of caretaker genes in that it cannot initiate tumors de novo, as illustrated here by the absence of medulloblastoma in Atoh1-Cre;Dicer1flox/flox mice. Furthermore, the endoribonuclease Dicer has recently been shown to maintain genomic stability in Schizosaccharomyces pombe by promoting transcription termination at sites associated with replication stress and DNA damage (Castel


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et al. 2014). This is likely to occur in higher eukaryotes. Therefore, we extrapolate that the tumor suppressor function of Dicer1 would be complex and would not involve the classical gatekeeper approach of directly regulating tumors by targeting effects that inhibit growth or promote cell death. We propose that more studies that characterize the genetic interplay between principal pathways, such as here, should be conducted, given that pathway interactions may produce nonadditive phenotypes that substantially differ from deregulation of a pathway in isolation. Here, we successfully demonstrated that the two principal pathways of miRNA biogenesis and ShhPtch signaling combinatorially and nonadditively influence the latency and severity of cerebellar hypoplasia and medulloblastoma. Novel insights were reached on the basis of these observations, such as a potential mode of action for Dicer1 haploinsufficiency in Ptch1-induced medulloblastoma, and indications that miRNAs and Shh-Ptch signaling together regulate an early developmental transition in granule cells.

Acknowledgments We thank Witold Filipowicz for the Dicer 349 antibody and for his intellectual input. Confocal imaging was performed in the Australian Cancer Research Foundation’s Imaging Facility at the Institute for Molecular Bioscience. This work was supported by the National Health and Medical Research Council of Australia.

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GENETICS Supporting Information www.genetics.org/lookup/suppl/doi:10.1534/genetics.115.184176/-/DC1

MicroRNA Biogenesis and Hedgehog-Patched Signaling Cooperate to Regulate an Important Developmental Transition in Granule Cell Development Lena Constantin, Myrna Constantin, and Brandon J. Wainwright

Copyright © 2016 by the Genetics Society of America DOI: 10.1534/genetics.115.184176

Table S1. Cause of death and days alive in Atoh1-Cre;Dicer1flox;Ptch1flox mice. e, euthanized at end of trial; h, hydrocephalus; H, hypoplasia; Hh, hypoplasia accompanied with hydrocephalus; MB, hyperplasia that manifested as medulloblastoma; O, other/unrelated.

Available for download as a .docx file at http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.115.184176//DC1/TableS1.docx